Carotenuto Lidia, Keminer Oliver, Carleo Giusy, Zaliani Andrea, Leo Antonio, Citraro Rita, De Sarro Giovambattista, Dirkx Nina, Kaji Marcus, Weckhuysen Sarah, Reinshagen Jeanette, Barrese Vincenzo, Guida Natascia, Ostacolo Carmine, Miceli Francesco, Gribbon Philip, Taglialatela Maurizio
Division of Pharmacology, Department of Neuroscience, University of Naples Federico II, Naples, Italy.
Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Discovery Research ScreeningPort, Hamburg, Germany.
Br J Pharmacol. 2025 Jul 23. doi: 10.1111/bph.70119.
Pharmacological activation of neuronal M-current mediated by Kv7 (Kv7.2-5) potassium channels is a validated mechanism for epilepsy treatment. However, since the market withdrawal of the prototype Kv7 activator retigabine, no Kv7 activator is clinically available for this condition. The object was to identify, characterise and validate new neuronal Kv7 channel activators for epilepsy treatment.
A fluorescence-based high-throughput assay was optimised in cells stably expressing Kv7 channels to screen two repurposing libraries including >8000 compounds. Whole-cell patch clamp, in silico docking, mutagenesis and multielectrode array recordings in human induced-pluripotent stem cell (hiPSCs)-derived cortical-like glutamatergic neurons (iNeurons) were used to evaluate compound(s) potency and efficacy, binding site, and effects on neuronal activity, respectively. Finally, anticonvulsant activity was assessed in two acute seizure models in male mice.
JNJ-37822681, a fast-dissociating D receptor antagonist in clinical development as antipsychotic, enhanced Kv7.2-5 currents with potency and efficacy largely comparable to retigabine. In Kv7.2 subunits, JNJ-37822681 binding site largely overlapped that for retigabine. In iNeurons, JNJ-37822681 enhanced the M-current, hyperpolarised the resting membrane potential and reduced spontaneous action potential firing. These effects were blocked by the Kv7 antagonist, XE-991, and were not reproduced by the D antagonist (-)-sulpiride. Finally, JNJ-37822681 showed anticonvulsant activity in two well-validated mouse models of acute seizures.
Our study reveals that JNJ-37822681, which lacks retigabine's potential safety issues due to chemical liability and is already confirmed as safe for human use, represents a potential treatment of Kv7-related neuronal hyperexcitability disorders.
由Kv7（Kv7.2 - 5）钾通道介导的神经元M电流的药理学激活是一种经过验证的癫痫治疗机制。然而，自原型Kv7激活剂瑞替加滨退出市场以来，尚无Kv7激活剂可用于临床治疗这种疾病。目的是鉴定、表征和验证用于癫痫治疗的新型神经元Kv7通道激活剂。
在稳定表达Kv7通道的细胞中优化基于荧光的高通量检测方法，以筛选两个包含8000多种化合物的重新利用文库。采用全细胞膜片钳、计算机模拟对接、诱变以及在人诱导多能干细胞（hiPSC）衍生的皮质样谷氨酸能神经元（iNeurons）中的多电极阵列记录，分别评估化合物的效力和功效、结合位点以及对神经元活动的影响。最后，在雄性小鼠的两种急性癫痫发作模型中评估抗惊厥活性。
JNJ - 37822681是一种正在临床开发中作为抗精神病药物的快速解离D受体拮抗剂，它增强Kv7.2 - 5电流的效力和功效与瑞替加滨大致相当。在Kv7.2亚基中，JNJ - 37822681的结合位点与瑞替加滨的结合位点大部分重叠。在iNeurons中，JNJ - 37822681增强M电流，使静息膜电位超极化并减少自发动作电位发放。这些作用被Kv7拮抗剂XE - 991阻断，而D受体拮抗剂（ - ）舒必利未重现这些作用。最后，JNJ - 37822681在两种经过充分验证的急性癫痫发作小鼠模型中显示出抗惊厥活性。
我们的研究表明，JNJ - 37822681由于化学特性不存在瑞替加滨潜在的安全问题，且已被证实对人体使用安全，它代表了一种治疗Kv7相关神经元兴奋性过高疾病的潜在药物。
